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A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors
Published on: December 7, 2014
Preexistence and evolution of imatinib mesylate-resistant clones in chronic myelogenous leukemia detected by a
K-A Kreuzer1, P Le Coutre, O Landt
1Medizinische Klinik m.S. Hämatologie und Onkologie, Campus Virchow-Klinikum, Universitätsklinikum Charité der Humboldt-Universität zu Berlin, Germany. karl-anton.kreuzer@charite.de
Abstract:
Recently, various mutations within the Abl sequence have been described that negatively affect imatinib binding to Bcr/Abl resulting in cellular resistance of chronic myeloid leukemia (CML) cells. So far, little is known as to whether these mutations are preexisting or develop under imatinib therapy as current mutation analyses are limited by a low sensitivity of approximately 1:2 (50%) to 1:5 (20%). By combining peptide nucleic acid (PNA)-based DNA clamping with a fluorescence hybridization probe assay, we developed a new and highly sensitive technique for the detection of known mutations within the Bcr/Abl kinase domain. With this approach we investigated 19 cases of CML refractory to imatinib treatment before and during therapy. By clamping of wild-type Abl through PNA we could effectively enhance the detection sensitivity for the Bcr/Abl mutations Thr315Ile, Glu255Lys, and Tyr253His such that 1 mutant cDNA molecule could be detected in 500 negatives (0.2%). We observed in one case that a Gly255Lys mutation was detectable before treatment. By DNA analysis of buccal swaps, a genetic polymorphism could be excluded. In two cases clonal evolution of known mutations developed gradually under treatment. In another case an initially detectable Tyr253His mutation disappeared after therapy onset but was again observed after 6 weeks of imatinib treatment. Preexisting and evolving Bcr/Abl mutations associated with an unfavorable prognosis could be safely detected by the presented technique. This may facilitate risk stratification in CML and may serve as a model for individualized molecular monitoring and therapeutic strategies in other malignant diseases.
Insights
A new highly sensitive method detects Bcr/Abl mutations in chronic myeloid leukemia (CML) cells, identifying preexisting and evolving mutations that confer imatinib resistance. This aids in risk stratification and personalized therapy for CML patients.
Area of Science:
- Hematology
- Molecular Biology
- Genetics
Background:
- Imatinib resistance in chronic myeloid leukemia (CML) is often caused by Bcr/Abl mutations.
- Current mutation detection methods lack the sensitivity to identify low-frequency mutations, hindering understanding of their origin (preexisting vs. acquired).
Purpose of the Study:
- To develop a highly sensitive assay for detecting known Bcr/Abl mutations in CML.
- To investigate the presence and evolution of these mutations in imatinib-refractory CML patients before and during therapy.
Main Methods:
- Combined peptide nucleic acid (PNA)-based DNA clamping with fluorescence hybridization probe assay.
- Achieved a detection sensitivity of 0.2% (1 mutant cDNA molecule in 500 negatives).
- Analyzed 19 imatinib-refractory CML cases before and during treatment.
Main Results:
- Detected preexisting mutations in one case before treatment.
- Observed gradual clonal evolution of mutations under therapy in two cases.
- Monitored dynamic changes in mutation presence during imatinib treatment.
Conclusions:
- The PNA-based assay safely detects preexisting and evolving Bcr/Abl mutations associated with poor prognosis in CML.
- This technique can facilitate risk stratification and individualized molecular monitoring in CML.
- The approach may serve as a model for monitoring other malignant diseases.
